Ceraul, Shane M.

Hynes, Wayne L.

Sonenshine, Daniel E.

Todd, Michelle

English

Old Dominion University

Old Dominion UniversityODU Digital CommonsBiological Sciences Faculty Publications Biological Sciences2005Defensin in Ticks: Molecular Characterization,Expression, and Role in Innate ImmunityDaniel E. SonenshineOld Dominion University, dsonensh@odu.eduWayne L. HynesOld Dominion University, whynes@odu.eduShane M. CeraulOld Dominion UniversityMichelle ToddFollow this and additional works at: https://digitalcommons.odu.edu/biology_fac_pubsPart of the Entomology Commons, Immunity Commons, and the Parasitology CommonsThis Presentation is brought to you for free and open access by the Biological Sciences at ODU Digital Commons. It has been accepted for inclusion inBiological Sciences Faculty Publications by an authorized administrator of ODU Digital Commons. For more information, please contactdigitalcommons@odu.edu.Repository CitationSonenshine, Daniel E.; Hynes, Wayne L.; Ceraul, Shane M.; and Todd, Michelle, "Defensin in Ticks: Molecular Characterization,Expression, and Role in Innate Immunity" (2005). Biological Sciences Faculty Publications. 134.https://digitalcommons.odu.edu/biology_fac_pubs/134Original Publication CitationSonenshine, D., Hynes, W., Ceraul, S., & Todd, S.M. (2005). Defensin in ticks: Molecular characterization, expression, and role in innateimmunity. Paper presented at the 5th International Conference on Ticks and Tick-Borne Pathogens, August 29th-September 2nd,Neuchatel, Switzerland.Defensin in ticks: molecular characterization, expression and role in innate immunity Daniel E. Sonenshine, Wayne L. Hynes, Shane M. Ceraul and S. Michelle Todd Department of Biological Sciences, Old Dominion University, Norfolk, Virginia hoate lmmaae Pathway Signal transduction TOLL receptors r l ·r:· ~ v~11 e n~{ pr;~ · 1 o~· ::===:::::::=:::::=;::::=::::=:; 3-L· 8, PG etc Signal transduction C;ll--m~dfi.at;d r;3po1 3; Ph!gocytosis 8ohible l_~npr ! ~J~ Defensins, lysozyme, other AMPs - ~ectins, opsonizing proteins/peptides MATERIALS AND METHODS •!• Antimicrobial assays against Borrelia burgdorferi, other bacteria •:• Gel electrophoresis - Western blot using anti-defensin antiserum •!• mRNA isolation •!• RT-PCR with defensin primer n- for D. variabilis (F - 5' GACTGCGCTTTGAGACGACAAA 3' (R-5' AGAAAGCATAACCATTTTTAATATGCATTT 3') •!•RT .. PCR with def ens in primer for I. scapularis (F - 5' CGAAGGCAGAACACAGGTCAAGTA 3') (R- 5' GGAGTTTATTGAACAGCCGCCGGAAGGA 3') •!• 5' and 3' RACE with gene specific primers •!• Cloning and sequencing D. variabilis 1 hour Fate of B. burgdorferi spirochetes after injection into D. variabilis hemocoel L scapularis 1 hour Fate of B. burgdorferi spirochetes after injection into I. scapularis hemocoel 5 21.5 1 4 6 3 MW 2 lb 3 6h 4 5 24 h Solvent 'po P-she'.et Defensin: Structure •Primary structure contains 6 conserved cysteine residues •Tertiary structure-CSap motif -Central amphipathic alpha helix -Anti-parallel double stranded beta sheet -Disulfide bridges link alpha helix and beta sheet -Motif stabilized by 3 disulfide bonds ~<;--DEFENSIN: basics Cationic PI~ 8.5-9.3 •:• Most def ens ins 4 kDa ~p ides with at least 6 cysteines, active primarily against Gram + bacteria - also B. burgdorf eri •:• Def en sins ubiquitous plant and animal kingdoms •:• In insects, found fat body, hemocytes, mid gut •:• In ticks - primarily in hemocytes. --------------Defensin: Mode of action •Defensin binds to Binding negatively charged · 111 membrane accumula e~ •Defensin molecules accumulate •Membrane strains and result in pores permeabilizes membrane def ens in Natwe Reviews I Immunology Ganz. 2003. Nature Rev. lmmunol. 3:710-720 Defensi Hemocyte and . Fat body Lysates Peptide absent in midgut FB Hemo MW 1l'•.aLJ.J.1.-.1 u..r, u1 trypttc u1~e~t putative ue1e11~111 ua11u 1ru111 f ~ 1• hemocyte lysate 1885.7824 IO • 78 • • • • 971.3800 ! 988.4089 20 842.5100 I 10 1110.5782 1112.5929 1384.8898 MIN(mlz) 887.7890 1898.7970 1917.7593 2212.0855 ________ Submitted ____ Peptide sequence NCBI accession no. ID -~~~ -"--- --~ 1110.58 R)RGGYCSGIIK(Q) " " 1884.79 R)GFGCPLNQGACHNHCR(S) " " RESULTS - D. variabilis •!•Obtained 624 bp cDNA from hemocytes •!•Same from fat body and midgut •!•Sequencing 624 bp cDNA fragment revealed 225 bp ORF corresponding to a 7 4 amino acid preprodefensin •!• MRGLCICL VFLL VCGL VSAT AAAP AESEV AHL VRI\ GFG 'PLNQGA 'HNl/i 'RSIR-RRGG'Yl 'SGIIKQ1i TCYRN 1 - 32: Prepro region; 32 - 36: signal peptide 37 - 74: mature peptide (38 AA) 33 -----"---- ------~~--------~----~~~~"-------, RESULTS L scapularis Nucleotide and derived amino acid sequence of scapularisin, a defensin-like peptide in the tissues of the black-legged tick Ixodes scapularis A.ACA.AGCTTTTTNNNCCGGCACACTGCG.A.AGGCAG.A.ACACAGGTCAAGTAG.A.ACACTGCATCATC CCTTGA.AATC lM RV I AV TL I ALL VA GAF MTS 1 ATGAGGGTCATTGCTGTTACCTTGATCGCCCTTCTGGTTGCTGGAGCGTTTATGACTTCC 21 S A Q E E E N Q V A H V R V R R G F G C 61 AGCGCAC.A.AGAGG.A.AGA.AAACC.A.AGTGGCTCACGT GAGTTCGAC~~ GTTTTGGATGT 41 P F D Q G A C H R H C Q S I G R R G G Y 121 CCCTTCGACC.A.AGGGGCGTGTCACAGGCACTGCCAGAGCATCGGACGTCGCGGAGGTTAC 61 C A G F I K Q T C T C Y H N * 181 TGCGCGGGATTTATC.A.AGCAGACGTGCACATGCTACCAC.A.ACTAG 1 - 32 = Prepro region; Pink arrow = 33 - 36 signal sequence; red arrow = 37 - 7 4 mature peptide (38 AA). Consensus alignment Is MRVIAVTLIALLVAG~~TSSAQEEEN-QVAHVR G---------YG PFNQGA HRH QSIG-RRGGY AGFIKQT T YHN- 7~ Bm MRGIYICLXFVLXCGLVSGLADVPAES-EMAHL Dv MRGLCICLVFLLVCGLVSATAAAPAES-EVAHLR OmA MNKLFIVALVVALAVATMAQEVHNDVE-EQSVP QnB MNKLFIVALVVALAVATMAQEVHDDVE-EQSVP QnC MNKLFIVALVLALAVATMAHEVYDDVE-EPSVP QnD MNKLFIVALVLALAVATMAHEVHDDIE-EPSVP Ir MKVLAVSLAFLLIAGLISTSLAQNEEGGEKEL PFNQGA HRHCRSIR-RRGGY AGLIKQT T YRN- 87% ---------FG PLNQGACHNHCRSIR-RRGGY SGIIKQT T YRN- 79% ---------YGCPFNQYQCHSHCSGIRGYKGGYCKGTFKQT KCY--- 65% ---------YG PFNQYQ HSHCRGIRGYKGGY TGRFKQT KY--- 68% ---------YG PFNQYQCHSHCSGIRGYKGGY KGLFKQT NY--- 65% G---------FG PFNQYE HAHCSGVPGYKGGY KGLFKQT NY--- 62% GG--------YY PFFQDK HRHCRSFG-RKAGY GGFLKKT I VMK- 60% Ah MATVRNSRPEAAGEPSGVSSTEGDWRHIEKRDVSYQGEGNTRRFDNPFG PADEGK FDHCNNKA-YDIGY GGSYRATCV YRK- 39% g5flA MG MKAAFVLLVVGLCIMTDTA------------------G---------FG PNNYACHQHCKS IRGYCGGYCGWFRLR TCYR G - 60% Is = I. scapularis; Bm = B. microplus; Dv = D. variabilis; Om = Ornithodoros moubata; Ir = I. ricinus; Ah = Amblyomma hebraeum," MG = Mytilus galloprovincialis (mussel). •:•High degree variability among tick defensins •:•Phylogenetic analysis L ricinus complex, L scapularis and 1 ricinus are not sister taxa (Xu et al., 2003). • I r Soft tick ( 0. moubata) + ~ 0 . 4 -4266. 0 [M+Hj+ ----~---- ..... ---2000 4000 6000 eooo 10000 Ma-(mfz) + E I ~ 0 . 3 -1i e 0.2 - -------- --- - ---------------i 0 . 1 - --i a-l 15 5 [ 0Activl'>' (nvn) 20 Retention time (min) 25 25-~ ..9,! ·c: 20 '2 _g Cl) ::j_ -, { e ent? Hard tick (D. variabilis) --111CO 1000 7SO I oi tlOO Ll ! :mo ; -250 ~ I ~ ~~ ~ 10 15 ~ I N ~,p (-) 311 -«> TROPSA - midgut receptor for OspA B. burgdorferi spirochetes kDa .5 1 2 3 4 5 6 7 8 1 = Hemocyte lysate; 2 = Midgut no challenge; 3 = midgut Bb; 4 = MW markers; 5 = Midgut peptidoglycan; 6 = midgut 5% hemoglobin; 7 = midgut rabbit serum; 8 = hemoglobin standard '------~_Arrow = defensin confirmed b try tic digestion/mass s ectrometry. ~-~ 2 3 kDa 14.5 8.0 6.0 3.5 : ..... !I 4 5 6 7 8 9 no C18 Sep Paks. 2, 30o/o ACN; 3, 40°/o ACN; 4, 70°/o ACN; 5, MW stds; ~~ 6, 30°/o ACN; 7, 40o/o ACN; 8, 80°/o ACN; 9, crude midgut extract; 10, rabbit serum; sample loading 10 pg/lane. Red bands submitted for analysis. Table 1. Results of tryptic digestion/mass spectrometry analysis for identification of peptides recovered from gel slices of protein gels. A Hemocytes rMW band Hemocyte lysate 4.95 band Representative partial amino acid sequences found GFGCPLNQGACHNHCRS Mass 1884.8 Identification Tick defensin (varisin) Native MW 4229.0 ·---~---------------------------B M.d t . 1 .sru 5.01- AVGHLDDLPGALSTLSDHAHK I 2267.2 a -chain I 15482.5 5.62 kDa I hemoglobin I band I -" VLAAFSEGLSHLDNLK I 1713.9 P-chain 16141.5 I l hemoglobin ·1 I " TITLEVEPSDTIENVK 1787.9 Ubiquitin I 17959.7 -I +-14-16 I FLANVSTVL TSK 1279.7 a -chain I 15482.5 I kDaband I hemoglobin I I ----·-·· t«----" VVAGVANALAHK 1149.7 · P-chain 6141.5 hemoglobin -" AADETWEPFASGK 1408.6 Transthyretin 14000.0 I If!( SUMMARY D. variabilis •!• Preprodefensin produced/stored in hemocytes; mature peptide secreted in response to microbial challenge, e.g., B. b11rgilorferi •!• Sequencing generated 624 bp amplicon; 225 bp translates 74 AA preprodefensin, 38 AA mature peptide; only 1 isoform. •!• Midgut - transcript found but no peptide; defensins (multiple isoforms) found in soft ticks. •!• Small peptides (from 5 - 11 kDa) are digestive fragments of a-chain, 6-chain hemoglobin, ubiquitin and transtherytin. SUMMARY (continued) I. scapularis •!• Sequencing generated 410 hp amplicon; 225 hp translates 74 AA preprodefensin, 38 AA mature peptide, 1 isoform •!• /. scap11/aris similarity to D. variabilis 79°/o; to/. ricin11s 60°/o. •!• Transcript present in different tissues •!• No evidence peptide - not upregulated in response to microbial challenge FUTURE RESEARCH !• D. variabilis: •!• How do hemocytes recognize - regulate expression of defensin. Role of TOLL /IMD ( other?) regulatory pathways. •!• How does tick midgut control invading microbes? •!• How do tick-borne pathogenic microbes escape destruction and colonize tick tissues? !• I. scapularis: •!• Defensin transcript expressed; why no peptide? Role in innate immunity? !• Other tick species? •!• Hebraein (A1nblyo1111na /1ebrae11111) and other novel antimicrobial peptides 

Defensin in Ticks: Molecular Characterization, Expression, and Role in Innate Immunity

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Defensin in Ticks: Molecular Characterization, Expression, and Role in Innate Immunity

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